This proposal aims at deciphering the mechanism by which the newly discovered Misshapen-Warts-Yorkie pathway acts in the epithelial niche to regulate intestinal stem cell (ISC) division in Drosophila. Tissue stem cells, as well as cancer stem cells, can transition into higher proliferative states depending on the environment but the mechanism is largely unknown. Although mammalian ISC biology has gained tremendous progress in recently years, the complexity of the mammalian gastrointestinal tract posts strong barriers to understand niche regulation of ISC division. The Drosophila midgut has a relatively simple cellular organization, and midgut ISCs are the only mitotic cells that replenish the different cell types in this tissue under normal and pathogenic conditions. We have uncovered a novel role of the Ste20 kinase Misshapen functioning in differentiating precursor cells called enteroblasts to influence ISC division. Misshapen interacts with Warts to negatively regulate the transcriptional coactivator Yorkie, which promotes the production of the JAK-STAT pathway ligand Upd3 to increase ISC proliferation. We have also found that the mammalian homologue MAP4K4 interacts with LATS to inhibit YAP. Therefore, the Drosophila Misshapen-Warts-Yorkie and mammalian MAP4K4- LATS-YAP represent a new, conserved mechanism to regulate tissue homeostasis. The specific aims of this proposal are: 1. Determine the nature of the Misshapen-Warts signaling complex. We will test whether Misshapen directly phosphorylates Warts as a substrate and whether the Misshapen-Warts complex consists of other signaling components. 2. Determine whether the upstream kinase Tao1 activates Misshapen via phosphorylation at the threonine 194 residue. Our preliminary results show that T194 of Misshapen is phosphorylated and experiments are designed to prove that T194 phosphorylation by Tao1, or other kinases, is essential for intestinal homeostasis. 3. Investigate the physiologica processes that regulate Misshapen activity in enteroblasts. We will test the hypothesis that the physical interaction between enteroblasts and neighboring cells affects Misshapen activity, which in turn regulates the production of the appropriate amount of Upd3 for optimal intestinal tissue maintenance. The successful accomplishment of these aims will unveil some of the mechanisms by which the epithelial niche regulates ISC division and should provide insights into therapeutic strategies for human GI inflammatory diseases and cancer.